Method of performing traceless knockout and integration on gene of Acidithiobacillus caldus

Abstract

The invention discloses a method of performing traceless knockout on a chromogene or inserting an exogenous gene in a chromosome of Acidithiobacillus caldus. The method comprises the following steps: transferring a homologous recombinant plasmid containing plentiful restriction enzyme digestion sites of endonuclease I-SceI into the Acidithiobacillus caldus in a conjugation transfer manner, simultaneously, directionally inserting the homologous recombinant plasmid into the chromosome of Acidithiobacillus caldus by use of a homologous recombinant system of a cell itself, transferring a screenedsingle crossover to an I-SceI expression plasmid by an electroporation method, performing secondary homologous recombination on a single recon chromosome under a stress that the I-SceI enzyme cuts the chromosome, and optimizing and screening a double crossover bacterial strain to realize knockout and insertion of the gene. The invention also constructs a suicide type plasmid vector pSDUDI for homologous recombination and a plasmid pSDU1-I-SceI for expressing the I-SceI enzyme effectively, and provides convenience for the traceless knockout and the integration of the chromogene in the Acidithiobacillus caldus. The method provided by the invention provides a new way for intensive study on and modification of Acidithiobacillus caldus.

Description

technical field [0001] The invention relates to a gene traceless knockout and integration method of acidophilic Thiobacillus acidothermophilus, in particular to a method for seamlessly knocking out the chromosome gene of acidophilic Thiobacillus thermophilic or inserting an exogenous gene into the chromosome of acidophilus Thiobacillus thermophilic method, and a suicide plasmid vector pSDUDI for homologous recombination in chromosomal gene scarless knockout and integration, and a plasmid pSDU1-I- expressing meganuclease in Thiobacillus acidophilus. SceI. Background technique [0002] Acidithiobacillus caduls (A.caldus) is a kind of Gram-negative obligate autotrophic sulfur-oxidizing bacteria, which plays a very important role in bacterial leaching and can significantly increase the iron-oxidizing bacteria such as Acidithiobacillus The leaching efficiency of ferroxidans, Leptospirillum ferrooxidans, etc. In recent years, studies have found that A. can improve the ore leachi...

AI Technical Summary

Problems solved by technology

Gene knockout and integration are important methods for in-depth study of bacterial gene function, bacterial growth metabolism and bacterial molecular transformation. Rawlings' laboratory reported that the arsB and tetH genes of A. caldus were replaced by the A. caldus homologous recombination system Km gene, so as to achieve the purpose of gene knockout [4] (Leonardo J.van Zyl, Jolanda M.van Munster, and Douglas E.Rawlings., Appl Environ Microbiol., 2008, Vol.74, No.18, 5686-5694), but the efficiency of this knockout method is unstable, One arsB knockout strain was screened from 23 strains, and one tetH knockout strain was screened from 249 strains, and this knockout method needs to introduce an antibiotic resistance gene for every gene knockout, given that in A The number of effective antibiotics that .caldus can use is limited, so this knockout method cannot knockout multiple genes in the same strain, and there is no report of gene knockout in A.caldus

Therefore, there is no report on the method of efficiently knocking out the A.caldus gene or inserting the foreign gene into the chromosome of A.caldus in the world.

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